1. Field of the Invention
The present invention relates generally to the cleavage of β-amyloid precursor protein to produce β-amyloid peptide. More particularly, the present invention relates to isolated and purified compositions containing an enzyme responsible for such cleavage (β-secretase) and assays for identifying inhibitors of β-secretase.
Alzheimer's disease is characterized by the presence of numerous amyloid plaques and neurofibrillary tangles (highly insoluble protein aggregates) present in the brains of Alzheimer's disease patients, particularly in those regions involved with memory and cognition. β-amyloid peptide is a major constituent of amyloid plaque which is produced by cleavage of β-amyloid precursor protein. It is presently believed that a normal (non-pathogenic) processing of the β-amyloid precursor protein occurs via cleavage by a putative “α-secretase” which cleaves between amino acids 16 and 17 of the β-amyloid peptide region within the protein. It is further believed that pathogenic processing occurs in part via a putative “β-secretase” which cleaves at the amino-terminus of the β-amyloid peptide region within the precursor protein. Heretofore, however, the existence of β-secretase has not been confirmed.
The identification, isolation, and characterization of novel biological molecules having unique activities is generally useful. For example, novel enzymes can be used to catalyze reactions of a type associated with their class. In particular, novel proteases can be used to cleave proteins for a variety of purposes, and the availability of new proteases provides unique capabilities. In addition to such uses associated with enzymes in general, the identification, isolation, and purification of the putative β-secretase enzyme would permit chemical modeling of a critical event in the pathology of Alzheimer's disease and would allow the screening of compounds to determine their ability to inhibit β-secretase activity.
For these reasons, it would be desirable to isolate, purify, and characterize the enzyme responsible for the pathogenic cleavage of β-amyloid precursor protein at the amino-terminus of the β-amyloid peptide region. In particular, it would be desirable to utilize such an enzyme (referred to hereinafter as β-secretase) in methods for screening candidate drugs for the ability to inhibit the activity of β-secretase in in vitro systems. It would be particularly desirable if such screening assays could be performed in a rapid format which would permit the screening of large numbers of test drugs in automated fashion.
2. Description of the Background Art
β-amyloid precursor protein (APP) is expressed in three differently-spliced forms of 695, 751, and 770 amino acids, and “normal” processing involves proteolytic cleavage at a site between residues Lys16 and Leu17 in the β-amyloid peptide. Kang et al. (1987) Nature 325:773-776. Soluble β-amyloid peptide which has been cleaved at the putative β-secretase site has also been found in the culture medium of non-diseased cells (Haass et al. (1992) Nature 359:322-325) and in CSF from healthy humans and animals (Seubert et al. (1992) Nature 359:325-327). The possible existence of the putative β-secretase is discussed in, for example, Selkoe, “Cell Biology of the Amyloid β-Protein and the Mechanism of Alzheimer's Disease,” in Annual Review of Cell Biology, Spudich et al., eds., Annual Review, Inc., Palo Alto, Calif., vol. 10, 1994. The Swedish mutation of APP is also discussed in Selkoe, supra. See also, Esch et al. (1994) Science 248:1122.